Ionizing Radiation kills cancer cells by generating DNA damage. Resistance to Ionizing Radiation is generally determined by DNA repair mechanisms that can repair radiation-induced DNA damage. The most toxic effect of radiation is represented by the formation of double stranded DNA breaks. These breaks can be repaired by Homologous Recombination, and Non-Homologous End Joining DNA repair mechanisms. Inhibition of these mechanisms results in increased radiosensitivity since repair of radiation-induced double strand breaks is blocked. Thus, understanding the molecular mechanisms of double strand break repair is essential for designing novel radiosensitization therapies. We have recently uncovered a novel mechanism that regulates radioresistance, centered on the protein PARP14. PARP14 is a mono-ADP-ribosyltransferase, which unlike its well-known cousin PARP1 is unable to catalyze poly-ADP-ribose chain formation but can only transfer a single molecule of ADP-ribose to substrates. Mono-ADP-ribosylation is a still mysterious post-translational modification, and its functions in the cell are poorly characterized. Or preliminary results presented here show that PARP14 promotes repair of double strand breaks by activating Homologous Recombination DNA repair. Moreover, our preliminary results presented here indicate a mechanism for this activation: PARP14 mono-ADP-ribosylates the recombination factor RAD51 to promote RAD51 removal from D-loops thus allowing D-loop extension past the break and complete the recombination reaction. Thus, our work shows that PARP14 inhibition results in radiosensitivity by downregulating Homologous Recombination-dependent repair of radiation-induced double strand DNA breaks. In this proposal, we test the hypothesis that PARP14 promotes the repair of radiation-induced double strand DNA breaks by Homologous Recombination, through RAD51 mono-ADP-ribosylation. We will employ a comprehensive, integrative approach to study: 1) the impact of PARP14 on the repair of radiation-induced DNA damage by Homologous Recombination and other DNA repair mechanisms, and 2) the role of RAD51 mono- ADP-ribosylation by PARP14 in repair of radiation-induced double strand breaks. Our study is highly innovative since it addresses a novel, previously unrecognized function of mono- ADP-ribosylation in radioresistance. Our work will unravel how mono-ADP-ribosylation by PARP14 promotes radiation resistance, and describe novel radiosensitization approaches, based on PARP14 inhibition.